α-Dystroglycan (α-DG) was recently identified as a receptor for lymphocytic choriomeningitis virus (LCMV) and several other arenaviruses, including Lassa fever virus (W. Cao, M. D. Henry, P. Borrow, H. Yamada, J. H. Elder, E. V. Ravkov, S. T. Nichol, R. W. Compans, K. P. Campbell, and M. B. A. Oldstone, Science 282:2079–2081, 1998). Data presented in this paper indicate that the affinity of binding of LCMV to α-DG determines viral tropism and the outcome of infection in mice. To characterize this relationship, we evaluated the interaction between α-DG and several LCMV strains, variants, and reassortants. These viruses could be divided into two groups with respect to affinity of binding to α-DG, dependence on this protein for cell entry, viral tropism, and disease course. Viruses that exhibited high-affinity binding to α-DG displayed a marked dependence on α-DG for cell entry and were blocked from infecting mouse 3T6 fibroblasts by 1 to 4 nM soluble α-DG. In addition, high-affinity binding to α-DG correlated with an ability to infiltrate the white pulp (T-dependent) area of the spleen, cause ablation of the cytotoxic T-lymphocyte (CTL) response by day 7 postinfection, and establish a persistent infection. In contrast, viruses with a lower affinity of binding to α-DG were only partially inhibited from infecting α-DG^−/− embryonic stem cells and required a concentration of soluble α-DG higher than 100 nM to prevent infection of mouse 3T6 fibroblasts. These viruses that bound at low affinity were mainly restricted to the splenic red pulp, and the host generated an effective CTL response that rapidly cleared the infection. Reassortants of viruses that bound to α-DG at high and low affinities were used to map genes responsible for the differences described to the S RNA, containing the virus attachment protein glycoprotein 1.